The present invention relates to polypeptides which stimulate bone growth.
Understanding of issues related to bone growth and strength has progressed over the years, a summary being provided in international patent application No. PCT/CA 94/00144, published under international publication No. WO 94/20615 on Sep. 15, 1994.
Various approaches to treatment of diseases involving reduction of bone mass and accompanying disorders are exemplified in the patent literature. For example, U.S. Pat. No. 4,877,864, issued Oct. 31, 1989 describes human and bovine xe2x80x9cbone inductive factors.xe2x80x9d International patent application published Sep. 17, 1992 under No. 92/15815 describes a protein derived from a porcine pancreas which acts to depress serum calcium levels for treatment of bone disorders that cause elevation of serum calcium levels. European Patent Application No. 504 938 published Sep. 23, 1992 describes the use of di- or tripeptides which inhibit cysteine protease in the treatment of bone diseases. International patent application published Sep. 3, 1992 under No. 92/14481 discloses a composition for inducing bone grow the composition containing activin and bone morphogenic protein. European Patent Application No. 499 242 published Aug. 19, 1992 describes the use of cell growth factor compositions thought to be useful in bone diseases involving bone mass reduction because they cause osteoblast proliferation. International patent application published Jun. 25, 1992 under No. 92/10515 1992 describes a drug containing the human N-terminal parathyroid hormone (PTH) fragment 1-37. European Patent Application No. 451 867 published Sep. 16, 1991 describes parathyroid hormone peptide antagonists for treating dysbolism associated with calcium or phosphoric acid, such as osteoporosis. U.S. Pat. No. 5,461,034 issued Oct. 24, 1995 to Yissum Research Development Company of the Hebrew University of Jerusalem describes osteogenic growth polypeptides identified from regenerating bone marrow.
A relatively short half life of PTH in the blood serum and the positive effect of intermittent PTH injection on bone volume led the present investigator to the hypothesis that PTH may in some way lead to induction of a second factor into the circulatory system. The presence of such a second factor in blood serum of rats and of humans has thus been investigated.
It has been found possible to isolate from rat blood serum a polypeptide substance which, upon administration to rats incapable of producing PTH (parathyroidectomized rats), produces an increase in the observed bone mineral apposition rate. A nucleic acid probe, based on the amino acid sequence of the rat peptide was synthesized and used to screen a human liver cDNA fetal library in order to isolate a human nucleic acid sequence coding for a human bone apposition polypeptide. A polypeptide derived from the nucleic acid sequence was thus chemically synthesized according to the derived sequence Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Pro (SEQ ID NO:1). It has been observed that the bone apposition rate in intact rats increases in a dose dependent fashion upon administration of this chemically synthesized compound. Reduced bone growth, normally observed for ovariectomized rats, was observed not to occur in rats after being administered with the polypeptide over a four week period beginning two weeks after ovariectomization. Bone calcium density was found to be maintained in ovariectomized rats administered with the polypeptide over an eight week period beginning eight weeks after ovariectomization.
It is thought possible that the active polypeptide is a dimer of the foregoing sequence, there being evidence of significant dimer formation, presumably due to a disulfide bridge between two polypeptides having the sequence shown.
A modified form of the polypeptide containing a cysxe2x86x92ala substitution was thus synthesized: Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Ala Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Pro (SEQ ID NO:3). Some of the bone stimulatory effects of the xe2x80x9cnormalxe2x80x9d polypeptide (SEQ ID NO:1) were found for the modified polypeptide.
In other experiments, the bone mineral apposition rate in rats administered with rabbit antibodies to the normal polypeptide (SEQ ID NO:1) was found to be suppressed. The suppression was found to be attenuated in rats administered with both the normal polypeptide and antibodies to same.
Further, certain polypeptide fragments of the normal polypeptide (SEQ ID NO:1) have been synthesized and each has been found to have bone stimulatory effects:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Valxe2x80x83xe2x80x83SEQ ID NO:4:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Alaxe2x80x83xe2x80x83SEQ ID NO:5:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leuxe2x80x83xe2x80x83SEQ ID NO:6:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ilexe2x80x83xe2x80x83SEQ ID NO:7:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lysxe2x80x83xe2x80x83SEQ ID NO:8:
xe2x80x83Arg Thr Asn Glu His Thr Ala Asp Cys Lysxe2x80x83xe2x80x83SEQ ID NO:9:
Further, the polypeptide identified as SEQ ID NO: 7 has been found to increase bone calcium content of ovariectomized rats when administered over a period of eight or twelve weeks.
Other polypeptide fragments of the normal polypeptide (SEQ ID NO: 1) have also been synthesized and have been found to lack the bone stimulatory effect found for the normal polypeptide:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Glnxe2x80x83xe2x80x83SEQ ID NO:10:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asnxe2x80x83xe2x80x83SEQ ID NO:11:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Glnxe2x80x83xe2x80x83SEQ ID NO:12:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Aspxe2x80x83xe2x80x83SEQ ID NO:13:
Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Aspxe2x80x83xe2x80x83SEQ ID NO:14:
Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asnxe2x80x83xe2x80x83SEQ ID NO:15:
Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ilexe2x80x83xe2x80x83SEQ ID NO:16:
The polypeptide identified as SEQ ID NO:9 was modified to include a protecting group at each end. The N-terminus was thus acetylated and C-terminus was amidated. The activity of this protected polypeptide, identified as SEQ ID NO:24, was found to increase the bone mineral apposition rate in rats beyond that observed for each of the polypeptides identified as SEQ ID NOs: 1,7 and 9.
It has been reported that histidine and cysteine residues can effect degradation of asparaginyl- and aspartyl-containing polypeptides in the absence of catalytic enzymes [Int. J. Peptide Protein Res. 45, 1995, 547,553]. The following analogues of the polypeptide identified as SEQ ID NO:9 were tested, for stability and for effects on bone mineral apposition rate:
CH3CO-Arg Thr Asn Glu His Thr Ala Glu Cys Lys-NH2xe2x80x83xe2x80x83SEQ ID NO:25
CH3CO-Arg Thr Gln Glu His Thr Ala Glu Cys Lys-NH2xe2x80x83xe2x80x83SEQ ID NO:26
xe2x80x83CH3CO-Arg Thr Gln Glu His Thr Ala Asp Cys Lys-NH2xe2x80x83xe2x80x83SEQ ID NO:27
In terms of stability under the various conditions tested, the polypeptides identified as SEQ ID NOs:25 and 26 were found to be more stable than those identified as SEQ ID NOs:9, 7 and 24. The polypeptide identified as SEQ ID NO:27 was found to be less stable than any of SEQ ID NOs: 7, 9, 24, 25 and 26.
Each of the polypeptides identified as SEQ ID NOs:24, 25, 26 and 27 were found to increase the bone apposition rate over that observed for control rats.
Polypeptide sequences corresponding to SEQ ID NOs:25, 26 and 27 in which the terminal amino acid residues are not protected are referred to herein as SEQ ID NOs:28, 29 and 30, respectively.
It has further been found, by a series of substitutions that the general charge pattern, based on the side chains of the component amino acids, is important to the activity of the 10-amino sequences identified as SEQ ID NOs:9, 24, 25 26 and 27:
⊕-{circumflex over (xc3x97)}-{circumflex over (xc3x97)}-xe2x8ax96-⊕-{circumflex over (xc3x97)}-{circumflex over (xc3x97)}-xe2x8ax96-cys-⊕
In the case of each side chain indicated by an xe2x80x9cXxe2x80x9d, the side chain does not bear a full ionic charge under physiological conditions. An equivalent way of describing this arrangement of charges within the polypeptide is to say that the polypeptide has an amino acid sequence of up to 10+q amino acids, wherein, under physiological conditions, residues numbered n, n+4, n+9 are positively charged amino acids, residues numbered n+3, n+7 are negatively charged amino acids, wherein the remaining amino acids are nonpolar amino acids or uncharged polar amino acids, and wherein n is an integer from 1 to 1+q. As polypeptides having between 10 and 36 amino acids have been shown to have bone stimulatory activity, q can be from 0 up to 26. In particular embodiments, q is up to 20, 15, 14, 10, and 5.
Particular sequences shown to retain bone stimulatory activity include those in which substitutions have been made as follows:
R-T/A-N/Q/A-E-H-T/A-G/A-E/D-C/Y/A/S-Kxe2x80x83xe2x80x83(SEQ ID NO:63)
As can be seen, each of the threonine residues in the second and sixth positions, respectively, of the sequence can be substituted by alanine. The oxygen atom contained in the side chain of threonine is generally considered to impart a negative polar charge to the side chain while the hydrocarbon side chain of alanine is generally considered to be non-polar. At the third position of the sequence, the asparagine residue has a side chain that contains the polar amide group and this can be interchanged with glutamine, having a similarly polar amide group in its side chain, or it can be interchanged with alanine having a non-polar side chain. In the case of the seventh position of the sequence, a glycine which has only a hydrogen radical as a side chain and which would not be considered to be polar, can be interchanged with the alanine residue. In the eighth position of the illustrated sequence, the aspartic acid residue can be replaced by a glutamic acid residue, both of which have side chains that include carboxylic acid groups, which under physiological conditions can be deprotonated and thus have a negative charge. In the ninth position, there is normally a cysteine residue which contains an xe2x80x9cSHxe2x80x9d group and this has been shown to react with the SH group of a second molecule resulting in a dimer. The cysteine residue can be substituted for by a tyrosine, alanine or serine with retention of bone-stimulatory activity of the compound.
Polypeptides in which alanine has been substituted in place of each of the ammo acids having a charged side chain, SEQ NOs:34, 35, 36, 37 and 38, were found to lack or to exhibit substantially less bone stimulatory activity than the family of compounds in which the full general charge pattern and spacing was retained. On the other band, sequences in which the second, third, sixth and seventh amino acids were substituted by alanine (or glycine in the case of the seventh amino acid, which is alanine in the parent sequence, SEQ ID NO:9), SEQ ID NOs:39, 40, 41 and 42, (encoded by SEQ ID NOs:55, 56, 57 and 58, respectively) largely retain bone stimulatory activity.
A polypeptide in which the ninth amino acid, cysteine, has been replaced by tyrosine (SEQ ID NO:43, coding SEQ ID NO:59) was found to have some bone stimulatory activity.
A polypeptide in which the third amino acid, asparagine, has been replaced by glutamine, the eighth amino acid, aspartic acid, has been replaced by glutaric acid, and the ninth amino acid, cysteine, has been replaced by tyrosine (SEQ ID NO:44, coding SEQ ID NO:60) was found to have bone stimulatory activity.
A polypeptide in which the third amino acid, asparagine, has been replaced by glutamine, the eighth amino acid, aspartic acid, has been replaced by glutaric acid, and the ninth amino acid, cysteine, has been replaced by tyrosine (SEQ ID NO:45, coding SEQ ID NO:61) was found to have bone stimulatory activity.
A polypeptide in which the third amino acid, asparagine, has been replaced by glutamine, the eighth amino acid, aspartic acid, has been replaced by glutamic acid, and the ninth amino acid, cysteine, has been replaced by serine (SEQ ID NO:46, coding SEQ ID NO:62) was found to have bone stipulatory activity.
In a particular aspect, for example, polypeptides identified as SEQ ID NOs:24, 25, 26, 27, 39, 40, 41, 42, 43, 44, 45 and 46 (corresponding to sequences lacking terminal modification, SEQ ID NOs:9, 28, 29, 30, 47, 48, 49, 50, 51, 52, 53, and 54, respectively) the charge pattern of the compound consists essentially of that provided by the amino acid sequence corresponding to SEQ ID NO:9, that is, it bears side chain charges in the order of and spaced as the amino acid side chains of SEQ ID NO:9 and does not include other amino acids. The invention includes a compound with substitutions of the sequence corresponding to SEQ ID NO:9 which retain bone stimulatory activity in mammals.
In one aspect, the invention is a compound that is a polypeptide and the sequence from which the polypeptide is derived consists of up to 25 consecutive amino acids selected from the sequence corresponding to SEQ ID NO:1 and includes the charge pattern provided by SEQ ID NO:9. Such a compound thus has a polypeptide sequence that xe2x80x9cconsists of up to 25 consecutive amino acids selected from the sequence corresponding to SEQ ID NO:1xe2x80x9d. The compound can have more than 25 amino acids, but no single portion (string of contiguous amino acid residues) of the entire sequence has more than 25 consecutive amino acids selected from the sequence corresponding to SEQ ID NO:1.
In another aspect, the invention includes a compound having bone stimulatory activity in mammals, in which the compound has the charge distribution of the side chain charges provided by the amino acid sequence corresponding to SEQ ID NO:9 and having up to about 83% sequence homology with the parent sequence identified as SEQ ID NO:1. The invention also includes a polypeptide having bone stimulatory activity in mammals in which the polypeptide has the charge distribution of the side chain charges provided by the amino acid sequence identified as SEQ ID NO:9 and including at least one non-conservative substitution at position number 2, 3, 6 or 9 of SEQ ID NO:9.
The present invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:1 with (a) from one to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:1 (b) one to about 22 amino acids deleted from the C-terminus of SEQ ID NO:1, or both (a) and (b); or a functionally equivalent homologue. Correspondingly, the invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:3 with (a) from one to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:3 (b) one to about 22 amino acids deleted from the C-terminus of SEQ ID NO:3, or both (a) and (b); or a functionally equivalent homologue. Sequence homology in polypeptides and proteins is understood to those skilled in the art, as discussed, for example in Molecular Cell Biology (H. Lodish, D. Baltimore, A. Berk, S. L Zipursky, P. Matsudaira and J. Damett, Scientific American Books, New York City. Third Edition, 1995). Likewise, the invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:4 with (a) up to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:4, (b) up to about 16 amino acids deleted from the C-terminus of SEQ ID NO:4, or both (a) and (b); or a functionally equivalent homologue. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:5 with (a) up to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:5, b) up to about 11 amino acids deleted from the C-terminus of SEQ ID NO:5, or both (a) and (b); or a functionally equivalent homologue. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:6 with (a) up to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:6, (b) up to about 5 amino acids deleted from the C-terminus of SEQ ID NO:6, or both (a) and (b); or a functionally equivalent homologue. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:7 with (a) up to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:7, (b) up to about 1 amino acids deleted from the C-terminus of SEQ ID NO:4, or both (a) and (b); or a functionally equivalent homologue. The invention also includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:8 with up to about four 4 amino acids deleted from the N-terminus or a functionally equivalent homologue. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:9 or a functionally equivalent homologue thereof.
The invention includes a polypeptide up to about 30 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:9 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 25 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:9 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
Alternatively, the invention includes a polypeptide of up to about 20 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:9 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms. The C-terminal and/or the N-terminal of any polypeptide of the invention can be protected, by conventional or other means.
The invention includes a polypeptide of up to about 15 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:9 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide about 10 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:9 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:24.
The invention includes a polypeptide up to about 30 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:26 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 25 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:28 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof it bound to two hydrogen atoms.
Alternatively, the invention includes a polypeptide of up to about 20 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:28 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 15 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:28 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide about 10 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:28 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxy group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:25.
The invention includes a polypeptide up to about 30 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:29 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 25 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:29 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
Alternatively, the invention includes a polypeptide of up to about 20 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:29 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 15 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:29 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide about 10 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:29 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:26.
The invention includes a polypeptide up to about 30 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:30 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 25 amino acids in length, comprising an amino acid sequence corresponding to SEQ ID NO:30 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
Alternatively, the invention includes a polypeptide of up to about 20 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:30 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide of up to about 15 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:30 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms.
The invention includes a polypeptide about 10 amino acids in length comprising an amino acid sequence corresponding to SEQ ID NO:30 or a functionally equivalent homologue thereof which promotes bone growth in mammals. The polypeptide can have a protected terminal amino group, or a protected terminal carboxyl group, or both. The N-terminal protecting group can be an acetyl group. The C-terminal can be protected by conversion of the carboxyl group to an amide group, in which for example, the amino nitrogen thereof is bound to two hydrogen atoms. The invention includes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:27.
Polypeptides of the present invention can be incorporated into larger polypeptide sequences in which there is repetition of active sequences in a single molecule.
The inventive polypeptide can be synthetic and the amino acid sequence can have a molecular weight in the range of from about 1000 to 4000.
The invention includes a polypeptide having a sequence of amino acids sufficiently duplicative of another, i.e., second polypeptide having an amino acid sequence corresponding to SEQ ID NO:1 (or SEQ ID NO:3) with (a) from one to about four 4 amino acids deleted from the N-terminus of SEQ ID NO:1 (or SEQ ID NO:3), (b) one to about 22 amino acids deleted from the C-terminus of SEQ ID NO:1 or (SEQ ID NO:3), or both (a) and (b), or a functionally equivalent homologue thereof, such that the polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide. The polypeptide can be up to about 30 amino acids in length, for example, and the sequence of that polypeptide can be repeated within a larger polypeptide, or contain other polypeptide sequences which are not by themselves stimulate bone growth. Such polypeptide could also be up to 25, 20, 15 or about 10 amino acids in length.
xe2x80x9cSequence identity or homologyxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared polypeptide sequences, for example, is occupied by the same amino acid (for example, if a position in each of two polypeptide molecules is an alanine residue, then the molecules are homologous or sequences are identical at that position. The percent of homology between two molecules or sequence identity between two sequences is a function of the number of such matching positions shared by the two sequences divided by the number of positions compared xc3x97100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the polypeptide sequences METLIA and MPTWIF share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. The alignment can be performed according to two methods, the Clustal method and the J. Hein method.
The Clustal algorithm (using software available from DNASTAR Inc., 1228 South Park Street, Madison, Wis., USA, 1994) is recommended for aligning sequences whose similarity might not necessarily be evolutionary. The algorithm is described by Higgins, D. G. et al. 1989. CABIOS 5:151. The same software programme provides for aligning sequences according to the Jotun Hein method, which is recommended for aligning sequences of highly evolved families that have clear evolutionary relationship. The algorithm is described by Hein, J. 1990. Methods in Enzymology 183:626. Programme default settings (standard parameters) can be used. In the case of weighting amino acid residues based on evolutionary substitution patterns, charge, structural and chemical similarity, the default PAM250 setting can be used. For protein alignments, the pairwise alignment parameters are Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5 can be used.
The phrase xe2x80x9cselectively hybridizing toxe2x80x9d refers to a nucleic acid probe that, under appropriate hybridization conditions, hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of DNA or RNA. xe2x80x9cComplementaryxe2x80x9d or xe2x80x9ctargetxe2x80x9d nucleic acid sequences refer to those nucleic acids that selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe""s length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., (ed.) Greene Publishing and Wiley-Interscience, New York (1987).
xe2x80x9cStringent hybridization conditionsxe2x80x9d takes on its common meaning to a person skilled in the art here. Appropriate stringency conditions which promote nucleic acid hybridization, for example, 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C. are known to those skilled in the art. The following examples are found in Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6: For 50 ml of a first suitable hybridization solution, mix together 24 ml formamide, 12 ml 20xc3x97SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml 100xc3x97Denhardt""s solution, 2.5 ml deionized H2O, 10 ml 50% dextran sulfate, and 0.5 ml 10% SDS. A second suitable hybridization solution can be 1% crystalline BSA (fraction V), 1 mM EDTA, 0.5 M Na2HPO4 pH 7.2, 7% SDS. The salt concentration in the wash step can be selected from a low stringency of about 2xc3x97SSC at 50xc2x0 C. to a high stringency of about 0.2xc3x97SSC at 50xc2x0 C. Both of these wash solutions may contain 0.1% SDS. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22xc2x0 C. to high stringency conditions, at about 65xc2x0 C. The cited reference gives more detail, but appropriate wash stringency depends on degree of homology and length of probe. If homology is 100%, a high temperature (65xc2x0 C. to 75xc2x0 C.) may be used. If homology is low, lower wash temperatures must be used. However, if the probe is very short ( less than 100 bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37xc2x0 C. to 40xc2x0 C.), and raises the temperature by 3-5xc2x0 C. intervals until background is low enough not to be a major factor in autoradiography.
In another aspect the invention is a synthetic polypeptide having in vivo bone stimulatory activity in mammals and which increases mineral content (i.e., calcium) in bones of mammals, having an amino acid sequence which is at least about 19% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 and having at least one amino acid deleted therefrom, or a functionally equivalent homologue.
The invention includes a synthetic polypeptide having in vivo bone stimulatory activity in mammals and which increases mineral content in bones of mammals, having an amino acid sequence which is at least about 22% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 and having at least one amino acid deleted therefrom.
The invention includes a synthetic polypeptide having in vivo bone stimulatory activity in mammals and which increases mineral content in bones of mammals, having an amino acid sequence which is at least about 25% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 and having at least one amino acid deleted therefrom.
The invention includes a synthetic polypeptide having in vivo bone stimulatory activity in mammals and which increases mineral content in bones of mammals, having an amino acid sequence which is at least about 28% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 and having at least one amino acid deleted therefrom.
The invention includes any of the foregoing synthetic polypeptides in which at least six amino acids deleted from the polypeptide sequence; or in which at least eleven amino acids deleted from the sequence; or in which at least sixteen amino acids deleted from the sequence; or in which at least twenty-one amino acids deleted from the sequence; or in which at least twenty-six amino acids deleted from the sequence.
The invention includes a polypeptide having a sequence of amino acids sufficiently duplicative of one of the foregoing synthetic polypeptides such that the polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the synthetic polypeptide.
In another aspect the invention is a polypeptide exhibiting bone stimulatory activity in mammals; the polypeptide having the sequence identified as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; or SEQ ID NO:30; analogues thereof wherein the amino acids in the sequence may be substituted, deleted or added, so long as the bone stimulatory activity in mammals derived the three dimensional structure of the sequence is preserved; and conjugates of each of the polypeptides or analogues thereof, wherein if the polypeptide sequence has that identified as SEQ ID NO:1, then there is at least one amino acid deleted therefrom. The invention includes a polypeptide that has a sequence of amino acids sufficiently duplicative of such a bone stimulatory polypeptide (or a functionally equivalent homologue thereof) that the polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the bone stimulatory polypeptide.
In another aspect, the invention is a polypeptide that includes an amino acid sequence that is between 19% and 900% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 86% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 69% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 56% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 42% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 39% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 28% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 90% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 86% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 69% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 56% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 42% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 39% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or a functionally equivalent homologue that has bone stimulatory activity in a mammal.
In another aspect, the invention is a polypeptide having bone stimulatory activity, the polypeptide comprising an amino acid sequence that has 10+q amino acids, wherein, under physiological conditions, residues numbered n, n+4, n+9 are positively charged amino acids, residues numbered n+3, n+7 are negatively charged amino acids, wherein the remaining amino acids are nonpolar amino acids or uncharged polar amino acids, wherein n is an integer from 1 to 1+q and q is an integer from 0 to 15; and, wherein the polypeptide includes no more than 25 consecutive amino acids corresponding to the amino acid sequence identified as SEQ ID NO:1.
A person skilled in the art would of course, understand that modifications of sequences, such as those identified as SEQ ID NO:1 or SEQ ID NO:9, described herein are to be each taken as though made separately and independently of each other.
In the case of this aspect of the invention, q can be 10, 5 or 0. The remaining amino acids can be selected from the group consisting of glycine, alanine, valine, isoleucine, serine, threonine, methionine, asparagine and glutamine. The residue numbered n+8 can be cysteine, tyrosine, alanine or serine; the residue numbered n can be arginine; the residue numbered n+1 can be alanine or threonine; the residue numbered n+2 can be alanine, asparagine, or glutamine; the residue numbered n+3 can be glutamic acid; the residue numbered n+4 can be histidine; the residue numbered n+5 can be threonine or alanine; the residue numbered n+8 can be glycine or alanine; the residue numbered n+7 can be glutamic acid or aspartic acid; and the residue numbered n+9 can be lysine.
The polypeptide can be a chimeric bone stimulating factor that includes any of the amino acid sequences described above as part of the invention.
The invention includes an agent for use in prevention and treatment of a bone reduction related disease that includes any polypeptide described above as part of the invention, including of course a chimeric polypeptide, as an active ingredient.
The invention is thus also a pharmaceutical composition for promoting bone growth, having a therapeutically effective amount of any polypeptide described above as part of the invention.
The invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide (or a pharmaceutical composition including the polypeptide) described above as part of the invention.
The invention includes the treatment of osteoporosis, promotion of bone growth in a mammal or treatment of a human of a bone reduction related disease.
The invention includes the use of a polypeptide having a sequence according to any polypeptide of the invention in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis, etc.
The invention includes a diagnostic kit or determining the presence of a polypeptide of the invention, in which the kit includes an antibody to a polypeptide (or polypeptides) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide (or polypeptides) and the antibody become bound together.
The invention includes an antibody which binds to a polypeptide of the invention. Particularly, the invention includes an antibody which binds to such a polypeptide when the antibody is synthesized using the polypeptide.
The invention includes molecules, such as isolated nucleotide sequences related to polypeptides of the invention. For example, the invention includes an isolated DNA fragment which encodes the expression of any of the polypeptides of the invention. It is of course understood that such fragments can vary from one another due to the degeneracy of the genetic code. Further, the invention includes a vector that has incorporated into it any such DNA sequence.
The invention includes an isolated DNA sequence encoding any amino acid sequence of the invention, or an analogue thereof, wherein the amino acids in the sequence may be substituted, deleted or added, so long as bone stimulatory activity in mammals derived from the three dimensional conformation of the sequence is preserved in a polypeptide having the amino acid sequence; sequences which hybridize to the DNA and encode an amino acid sequence of a polypeptide which displays bone stimulatory activity in mammals; and DNA which differs from the sequence due to the degeneracy of the genetic code.
The invention thus includes processes of producing any polypeptide of the invention, including a process which includes: a) preparing a DNA fragment containing a nucleotide sequence that encodes such a polypeptide; b) incorporating the DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication; c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide; and d) culturing the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture.